1. Field of the Invention
The present invention relates to a surface plasmon resonance (SPR) microscope using common-path phase-shift interferometry and, more particularly, to a microscope which combines SPR and modified common-path phase-shifting interferometry to measure the spatial phase variation caused by bio-molecular interactions upon a sensing chip.
2. Description of the Related Art
As shown in FIG. 1, a surface plasmon wave (SPW) is a physical phenomenon existing in the interference between a metal layer (an Au layer or an Ag layer in the visible light region) and a nonconductive dielectric medium (air or water). Free electrons between the metal film 12 and dielectric medium 13 have collective resonance oscillations along the interface excited by that incident light 14 which is coupled to the metal film 12 through a coupler 11 (a prism). This free electron oscillation is called the SPW.
A method of attenuated total reflection (ATR) is used to excite the free electrons to emit the non-radioactive SPW. That is, p-wave light parallel to the incident plane arrives in the dielectric medium 13 after the total internal reflection of the incident light 14 occurs thereon. The air 13 penetration depth of the p-wave is roughly a half of a wavelength, and therefore the incident light in the interference is also called an evanescent wave. The SPW fluctuates perpendicularly to the interference between the metal film 12 and dielectric medium 13, and it simultaneously propagates along the interference. As a result, and in addition to electromagnetic fields effectively concentrating in the interference, the electric field of the evanescent wave has a maximum value of intensity also existing therein. The intensity exponentially decreases in proportion to the distance from the interference. The SPR is an optical phenomenon in which incident P-wave light excites an SPW such that it reaches a resonance condition. Excitation of the SPR occurs when the wave vectors' parallel component of the incident light, kx, and the wave vector of the SPW, ksp, satisfy the following matching condition:kx=k0√{square root over (ε0)} sin θ=ksp;where θ is the incident angle of the light, k0=2 π/λ and ε0 is the wavelength dependent dielectric constant of the coupler 11. The wave vector of the SPW is regarded as a dispersion index and can be approximated by:
            k      sp        =                            k          0                ⁡                  (                                                    ɛ                1                            ⁢                              ɛ                2                                                                    ɛ                1                            +                              ɛ                2                                              )                            1        2              ;where ε1 and ε2 are the wavelength dependent complex dielectric constants of the metal film 12 and dielectric medium 13, respectively. When this matching condition is satisfied, most of the incident light energy is transferred to the surface plasmon, i.e. most of the incident light is absorbed by the excitation of the SPW. This phenomenon results in an attenuated reflected spectrum.
SPR biosensors can be applied to measure tiny variations in the dielectric constant or thickness of biomolecular materials at the interface without the need for additional labeling. The SPR technique has been widely applied to biomolecular interaction analysis (BIA). In addition to its inherent convenience, economy, and speed, on-going developments of the SPR technique are aimed at further enhancing its sensitivity, resolution and reliability in order to support the implementation of high-throughput screening processes.
Conventionally, SPR imaging systems apply a parallel monochromatic light beam oriented such that it incidents on a gold film through a prism or a grating-coupling. The angle of incidence is adjusted such that it is close to the SPR angle and the resulting SPR intensity pattern is detected by a CCD (charge coupled device) camera. Although this system has a high-throughput screening capability, its resolution is too low to permit the detection of biomolecules of low molecular weight or low concentration. Of the various SPR detector configurations, applying a prism-coupling to produce optical interference between the SPR phase and the reference light beam produces the best resolution. The current authors previously developed an SPR phase imaging system for the high-throughput real-time dynamic measurement of biomolecular interactions by detecting the variation in the dielectric constant or the thickness of the biomolecular material. However, in common with other SPR phase imaging systems, the developed system was unable to satisfy the strict demands of real-time BIA kinetic studies because it lacked long-term stability. Hence, the current study develops an SPR imaging system with long-term stability and high-resolution capabilities.